The industrial use of enzymes is often limited by their high cost and rapid inactivation. Soluble enzymes are lost with the product at the conclusion of a process, and must be replenished. One area of technological development involves modification of proteins to enhance their activity and/or stability. Processes, such as those involving site-directed mutagenesis and the cultivation of wild forms of enzymes in extreme environments, i.e., extremophiles, have led to significant advances in enzyme technology involving the reduction in the cost per unit of enzyme activity.
Another means to improve the economic feasibility of enzymes for industrial processes is through enzyme immobilization onto a matrix, which may facilitate re-use of the enzyme. Immobilization research has focused upon means to enhance the transfer of enzymes onto the support, and upon means to ensure that the immobilized enzymes remain active. Inactivation of enzymes during catalytic turnover is, however, a key obstacle which may limit the economic feasibility of enzyme-mediated processes. Enzymes may be inactivated by extremes of temperature, pH, shear, and also by free radicals and other reactive species present in the reaction medium. Immobilization technology has the potential to reduce such enzyme inactivation, and, thus, extend the enzyme's useful lifespan.
The development of an immobilized enzyme requires a choice of support matrix and a choice of enzyme immobilization method. These choices may have a dramatic impact on the quantity of enzyme transferred to the support, along with the activity and stability of the attached enzyme.
Activated charcoal is a well-known absorbent, and has been previously used for enzyme immobilization via absorption (A. S. Rani, M. L. M. Das, S. Satyanarayana, J. Mol. Catal. B. Enzymatic, 10, 471, 2000; W. Hassler, Purification with Activated Carbon, Chemical Publishing Co., New York, 1974), and following derivatization. Le Fevre and Saville, U.S. Pat. No. 5,998,183, describes the use of siliceous materials for enzyme immobilization.
Glutaraldehyde is a well-known protein cross-linking agent, used for enzyme immobilization and for fixation of samples for scanning electron microscopy (D. R. Walt and V. I. Agayn, Trends Anal. Chem., 13(10), 425, 1994). Its propensity to cause protein denaturation is well known. Typically, highly purified forms of glutaraldehyde are used for electron microscopy, whereas standard commercial grades of glutaraldehyde are typically used for immobilization. These commercial grades may include, in addition to the pure aldehyde, polymers of glutaraldehyde, cyclic structures, and acetals. A recent review (Walt and Agayn ibid) reached no clear consensus on the benefits/disadvantages of any of the forms of glutaraldehyde used for immobilization.
U.S. Pat. No. 4,438,196—Oreste J. Lantero Jr. is an example of derivation and describes immobilization onto activated carbon after the support has been derivatized using a polyamine compound or a copolymer of a polyamine and an epihalohydrin.